In silico discovery of non-psychoactive scaffolds in Cannabis halting SARS-CoV-2 host entry and replication machinery

Aim: Coronavirus disease still poses a global health threat which advocates continuous research efforts to develop effective therapeutics. Materials & methods: We screened out an array of 29 cannabis phytoligands for their viral spike-ACE2 complex and main protease (Mpro) inhibitory actions by in silico modeling to explore their possible dual viral entry and replication machinery inhibition. Physicochemical and pharmacokinetic parameters (ADMET) formulating drug-likeness were computed. Results: Among the studied phytoligands, cannabigerolic acid (2), cannabigerol (8), and its acid methyl ether (3) possessed the highest binding affinities to SARS-CoV-hACE2 complex essential for viral entry. Canniprene (24), cannabigerolic methyl ether (3) and cannabichromene (9) were the most promising Mpro inhibitors. Conclusion: These non-psychoactive cannabinoids could represent plausible therapeutics with added-prophylactic value as they halt both viral entry and replication machinery.


Viral replication inhibition
SARS-CoV-2 is the causative agent of raging pandemic of COVID-19 that is manifested by mild-to-severe respiratory tract inflammation leading to pneumonia, lung fibrosis and causing even death. Up till now there is no approved remedy for COVID-19, hence, many research endeavors have been directed toward finding therapeutic drug leads to combat the infection propagation. SARS-CoV-2 is a sense RNA virus bearing projections on its surface, i.e., envelope (E) and spike (S) glycoproteins with the latter initiating the infection via its attachment with host angiotensin-converting enzyme 2 (ACE2). ACE2 is an enzyme expressed in nasal and oral mucosa as well as lung and GI tract.
ACE2 (hACE2) and the coordinates of M pro in complex with its inhibitor 2-cyclohexyl-N-(3-pyridyl)acetamide were retrieved from the protein data bank (PDB ID 6LZG and 5R84, respectively) [11,[13][14][15]. The proteins were prepared employing the default "structure preparation" module settings, where hydrogen atoms were added, hydrogen bonds were optimized, atomic clashes were removed, and the crystal structures were refined. The docking protocol was adopted as previously reported in [16]. Docking results were presented as a list based on the S-scores with RMSD values below 2Å and graphically showing the phytoligands interactions.
In silico analysis of physicochemical properties & ADME profiling of the phytoligands The physicochemical properties and ADME profiles of the studied phytoligands in human body were predicted using SwissADME calculation toolkit [17] and the online-server PreADMET (https://preadmet.bmdrc.kr/) [18].

Results
Molecular docking analysis of cannabis phytoligands within SARS-CoV-2 spike (S) protein-ACE2 complex SARS-CoV-2 entry into the host cell is initiated by the spike (S) glycoprotein attachment [19] to hACE2 which proceeds by the cleavage of S protein by host cell proteases into S1 and S2 subunits for receptor recognition and cell membrane fusion, respectively. S1 is further subdivided into N-terminal domain (NTD) and C-terminal domain (CTD), which both function as a receptor-binding entity [20]. The S1 CTD of SARS-CoV-2 functions as the key region interacting with the hACE2 receptor. The crystal structure of SARS-CoV-2-CTD in complex with a single hACE2 molecule in asymmetric unit (PDB ID: 6LZG) was used in our study [13]. Docking simulations were used to explore the ability of the studied phytoligands in destabilizing the virus-enzyme complex or preventing its formation. We proposed that possible accommodation/fitting of such phytochemicals at the SARS-CoV-2-CTD-h2ACE interface and their possible interactions with the key amino acids of the complex may provide promising insights for the possible prophylactic action of the studied phytoligands.

Discussion
As revealed from molecular docking of the phytoligands within SARS-CoV-2 S protein-ACE2 complex, the most promising phytoligands were cannabigerolic acid (CBGA) (2) and its monomethyl ether derivative (3), and cannabigerol (8), belonging to cannabigerol-type phytocannabinoids which were featured by the presence of a linear isoprenyl residue. These phytochemicals were found to be most enriched in cannabis varieties resulted from hybridization [27]. They are nonpsychoactive with low cannabinoid (CB) receptors potency however, they possess powerful antioxidant and anti-inflammatory properties which make them good candidates for managing the inflammatory conditions [28]. Cannabichromene-type phytocannabinoids, represented by cannabichromene (9), possess isoprenyl moiety that is oxidatively fused to the resorcinyl ring. This type was reported to exhibit potent TRPA1 activation, anti-inflammatory and anti-nociceptive actions via the inhibition of cyclooxygenase enzyme and its associated prostaglandins but with no affinity to cannabinoid receptors, i.e., devoid of psychotropic activity [29]. These compounds are more abundant in the vegetative stage of hemp plant than in its reproductive stage [30]. Cannabifuran (25), an oxidatively cyclized analog of cannabinodiol (15), is commonly isolated from aged samples of hashish with no reported biological actions till now. The key residues involved in the complex formation are listed in bold. † The ligand-receptor complex binding free energy at RMSD ≤2Å.
Most of the promising phytoligands were able to accommodate into the interface and interact with the key aminoacids (Figures 1 & 2). Accordingly, they can destabilize or halt the virus-receptor engagement which is commonly dominated by polar contacts mediated by these key hydrophilic aminoacid residues [13]. Cannabigerolic acid monomethyl ether (3), cannabidiol (CBD) (6), cannabivarin (10), 9-tetrahydrocannabivarin ( 9 -THCV) (11), cannabinolic acid ( Cannabidiol (6) is a major phytocannabinoid in fiber hemp formed by spontaneous decarboxylation of its acidic form by the act of light and heat upon cannabis ageing. Despite having structural similarity with 9 -THC, it exhibits a distinct pharmacological profile and lacks any psychoactive properties with a very low affinity for cannabinoids receptors. Cannflavins A and B (22 & 23), methylated isoprenoid flavones, are two of unique C. sativa flavonoids with a well-reported anti-inflammatory action via the inhibition of 5-Lipoxygenase (5-LO) and prostaglandin E 2 synthase (mPGES-1) [9].
ACE2 inhibition was reported as being unfavorable in COVID-19 patients due to the consequent decrease in the production of angiotensin 1-7, that possess anti-inflammatory, antifibrotic and vasodilatory actions via the Mas Gly B496 Lys Tyr B453 Arg B403 Tyr B495 Lys B417 Tyr B453 Gly B496 Asn B501 Ser B494 Phe B497 Tyr B495 Lys A353 Tyr B505 Arg B403 Glu A37 Asp A38 Asn A33 His A34 Lys A353 Gly B496 Arg B403 His   Arg A393 Gly B496 Ser B494 Tyr B449 Lys A353 His A34 His A34 Asn A33 Lys B417 Gly B416 Lys B417 Arg B408 Gln A388 Tyr B453 Tyr B495  receptor [31]. The protective role of ACE2 was revealed in animal models of acute respiratory distress syndrome [32]. Patients who administer angiotensin-II inhibitors (ACE1) was reported to suffer from severe symptoms with a higher mortality rate than their counterparts who did not take these medications [33]. Thus, ligands not interacting with hACE2 side such as (+)-cannabichromenic acid (13) will be considered promising anti-SARS drug leads with a preventive potential. Canniprene (24), a unique dihydrostilbenoid to C. sativa, recorded the highest binding affinities to SARS-CoV-2 M pro , which possesses anti-inflammatory action via the inhibition of pro-inflammatory eicosanoids production. Canniprene was reported to exhibit more potent action than cannflavin A as 5-LO inhibitors, however, it is less effective in inhibiting mPGES-1 [34].
All the investigated phytoligands displayed acceptable aqueous solubility with canniprene (24) appearing at the top of the list with an excellent intestinal absorption (>90%), medium to high BBB penetration, moderate permeability through CaCo-2 cells model and low permeability through MDCK ones ( Table 3). Most of the phytoligands were predicted to possess moderate to high plasma proteins binding profiles and expected to be more bound to plasma proteins (PPB ≈ 96-100%). The investigated phytoligands were also predicted to be incapable of inhibiting cytochromes P450 2D6 (CYP2D6) but not CYP3A4, except cannabigerolic acid monomethyl ether (3) that was devoid of inhibiting both CYP2D6 and CYP3A4 (Table 3).
Indeed, oropharynx and nasopharynx are the main entry ports for SARS-CoV-2 as well as sources of transmission. Both saliva and nasopharyngeal secretions harbor a significant viral load in asymptomatic or presymptomatic virus carriers, and hence playing crucial role in both the pathogenicity and the transmission of SARS-CoV-2. Accordingly, an antiviral oral and nasopharyngeal rinses can be considered as one of the most efficient intervention in combating SARS-CoV-2 transmission [35]. Previous studies reported for the efficacy of mouthwashes in inhibiting viruses such as HIV, herpes simplex virus (HSV) and Middle East respiratory syndrome coronavirus (MERSCoV) [36][37][38]. Since, canniprene (24) possessed the best optimal ADMET profile, among the studied phytoligands, in addition to its most M pro inhibitory action, this phytoligand might serve as promising drug candidate to be administered orally as being devoid of any psychoactivity.
Cannabigerol-type phytocannabinoids, i.e., cannabigerolic acid (2), cannabigerol (8) and its acid methyl ether (3), possessed the most potent inhibitory action on the viral entry machinery in our in silico modeling study which makes them promising candidates to be administered via topical application in the form of mouthwash/gargles or intranasally. Cannabigerolic acid monomethyl ether (3) was revealed to exhibit a dual promising inhibitory action against both the viral entry and the replication machinery. However, the efficacy and tolerability of the identified phytoligands need to be confirmed for its effective integration into the clinical practice. Table 3.

Conclusion
There is an urgency in finding new anti-SARS therapies with an added prophylactic value to be employed side by side to the developed vaccines for an efficient curtailment of COVID-19. The current study posed new drug leads from cannabis with low toxicity and in silico dual inhibitory action against viral entry and replication machinery that is essential for a more efficient management of COVID-19. The phytoligands 'cannabigerolic acid (2), cannabigerolic acid monomethyl ether (3) and cannabichromene (9)' possessed the highest inhibitory potential against both viral entry and replication machineries as illustrated in the comparative histograms shown in Figure 3. It seems crucial now to develop dosage forms for local application in the oral cavity to halt the SARS-CoV-2 load for prophylaxis [44]. Intranasal delivery of the proposed drug leads is then suggested as an additional option to minimize COVID-19

Summary points
• In silico screening of 29 cannabis phytoligands against SARS spike-ACE2 complex and M pro .
• Cannabigerolic acid and cannabigerol were revealed as inhibitors of SARS-CoV-hACE2 complex.
• Canniprene and cannabigerolic methyl ether possessed anti-M pro activity.
• The identified phytoligands could serve as anti-SARS therapies with an added prophylactic value.

Author contributions
A Khattab: conceptualization, methodology, writing -original draft preparation; M Teleb: methodology, software, formal analysis, data curation. All authors read and approved the final manuscript.

Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript. In silico discovery of non-psychoactive scaffolds in Cannabis halting SARS-CoV-2

Availability of data & material
Data sharing is not applicable to this article because no datasets were generated or analyzed in this study.